SUPPLEMENTARY INFORMATION - Nature Research · KO-HFD, N=13. b, Insulin tolerance test after 6 hours fasting of male WT/dusp6-KO mice were fed on CD or HFD for 16 weeks. Data are

In the format provided by the authors and unedited.Nature Microbiology

Supplementary Information

Dual-specificity phosphatase 6 deficiency regulates gut microbiome and transcriptome response against diet-induced obesity in mice

Jhen-Wei Ruan, Sarah Statt, Chih-Ting Huang, Yi-Ting Tsai, Cheng-Chin Kuo, Hong-Lin Chan,Yu-Chieh Liao, Tse-Hua Tan, Cheng-Yuan Kao

© 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

SUPPLEMENTARY INFORMATIONARTICLE NUMBER: 16220 | DOI: 10.1038/NMICROBIOL.2016.220

NATURE MICROBIOLOGY | www.nature.com/naturemicrobiology 1

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Supplementary Figures

Supplementary Figure 1. Dusp6-deficiency increased glucose tolerance and alleviated

obesity-associated phenotypes in mice. a, Glucose tolerance test after 12 hours fasting of

male WT/dusp6 KO mice were fed on CD or HFD for 16 weeks. Data are presented as the

mean ± SEM of each time point. WT-CD, N=17; WT-HFD, N=15; dusp6 KO-CD, N=17; dusp6

KO-HFD, N=13. b, Insulin tolerance test after 6 hours fasting of male WT/dusp6-KO mice were

fed on CD or HFD for 16 weeks. Data are presented as the mean ± SEM of each time point.WT-CD,N=7;WT-HFD,N=7;dusp6KO-CD,N=5;dusp6KO-HFD,N=5.c, Blood glucose

level after 6 hours fasting of male WT/dusp6-KO mice fed on CD or HFD for 16 weeks. Data

are presented as the mean ± SEM. WT-CD, N=7; WT-HFD, N=7; dusp6 KO-CD, N=7; dusp6

KO-HFD, N=6. d, Weight of flank adipose tissue of male WT/dusp6-KO mice fed on CD or

HFD for 16 weeks. Data are presented as the mean ± SEM. WT-CD, N=10; WT-HFD, N=8;

dusp6 KO-CD, N=9; dusp6 KO-HFD, N=7. For (c) and (d), asterisks indicate statistical

difference (*P<0.05, ***P<0.0005) according to One-Way ANOVA analysis and Tukey

post-hoc test. e-f, Haematoxylin & eosin (H&E)-stained sections of flank adipose tissue (e) and

liver tissue (f) from male WT and dusp6-KO mice fed on CD or high-fat diet HFD for 16 weeks.

Scale bar, 100 µm.

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Supplementary Figure 2. Fecal/gut microbiome composition of FMT recipient mice. a,

Schematic diagram of fecal microbiome transplantation, metabolic tests and tissue sampling

of WT/D6KO microbiota recipient mice. b, Quantitative PCR validation of 16S rRNA gene of all

eubacteria before and after 16 weeks of HFD treatment. The plasmid contained 16S rRNA

sequence of Eubacteria was used as the standard to validate the absolute copy number of

each sample. The number of mice for each group: before HFD treatment, WT, N=18 and

dusp6KO, N=17; after 16 weeks of HFD treatment, WT, N=8 and dusp6KO, N=7. The red line

indicates the mean of each group. Double asterisks and triple asterisks indicate statistical

difference (P<0.005 and P<0.0005) of mean± SEM according to Mann-Whitney analysis. c-d,

Weighted-UniFrac (c) and Unweighted-UniFrac (d) Principle coordinates analysis (PCoA) plot

represents changes between chow diet (T0) and HFD (T16) fed wild-type and dusp6 knockout

mice. The number of mice per group: T0: WT-CD, N=11; dusp6 KO-CD, N=13; T16: WT-HFD,

N=6; dusp6 KO-HFD, N=6. e, Composition of Firmicutes family of WT/dusp6 KO microbiota

recipient mice before and after 16 weeks of HFD treatment. f, Composition of Proteobacteria

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family of WT/dusp6 KO microbiota recipient mice before and after 16 weeks of HFD treatment.

g, Cladogram generated from LEfSe analysis (α < 0.1 for factorial Kruskal-Wallis test),

showing the most differentially abundant microbial clades enriched in microbiota from

wild-type (green) or dusp6 knockout (red) FMT recipient mice fed HFD (T16). WT-HFD, N=8;

dusp6 KO-HFD, N=8. h,LDA scores of the differentially abundant microbial clades (with LDA

score >2 and significance of α < 0.1 determined by Kruskal-Wallis test) shown in (g). i, 5-6

weeks old male germ-free mice were oral gavaged with BHI broth culture of fecal microbiota

derived from representative donors and then fed on HFD for 16 weeks. Body weights were

monitored weekly. Data are presented as the mean ± SEM of each time point. CFMT-WT,

N=11; CFMT-dusp6 KO, N=12. j, Weight gain of WT/dusp6 KO CFMT recipient mice after 16

weeks of HFD treatment. Data are presented as the mean ± SEM. The asterisk indicates

statistical difference (P<0.05) according to Mann-Whitney analysis. CFMT-WT, N=11;

CFMT-dusp6 KO, N=12.

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Supplementary Figure 3. Transcriptome profile of CD or HFD treated WT/dusp6-KO

mice. a-b, Immuno-histochemistry analysis (a) and qRT-PCR analysis (b) of DUSP6 protein

or dusp6 gene expression in intestine of chow diet of HFD fed dusp6 KO mice.Scale bar, 100

µm. c-d, The number of overall DEGs (c) and gene ontology biological processes (d) that were

significantly enriched among up-regulated genes or down-regulated genes in the comparisons

of HFD-fed WT and dusp6-KO mice. e, The non-sense mutations of both alleles of DUSP6

gene of DUSP6 KO cells were confirmed by DNA sequencing analysis. (Green letters are not

included in gRNA but recognized by Cas9 protein). f, Immunoblot analysis of DUSP6 protein in

WT and DUSP6 KO Caco-2 cells. g, TEER analysis of WT and DUSP6 KO Caco-2 cells after

21 days of differentiation. Data are presented as the mean ± SEM (N=3 separate experiments).

The asterisk indicates statistical difference (P<0.05) according to two-tailed t-test. h, 2 hours

after oral gavaged with FITC conjugated 4kDa dextran, mouse serum was collected for

fluorescence measurement. Data are presented as the mean ± SEM. N=11 for each group.

The asterisk indicates statistical difference (P<0.05) according to Mann-Whitney analysis. i-j,

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qRT-PCR validations of the KEGG Ppar pathway (i) and tight junction pathway (j) that were

significantly enriched in the up-regulated genes of the comparison of dusp6-deficient mice to

wild-type mice in control diet in CD fed FMT recipient mice. Data are presented as the mean ±

SEM. N=8 for FMT-WT recipient mice and N=7 for FMT-Dusp6 KO recipient mice. The

asterisk indicates statistical difference (P<0.05) according to Mann-Whitney analysis. ns

indicates statistically non-significant.

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Supplementary Figure 4. KEGG immunological pathways down-regulated in HFD-fed

dusp6-deficient mice. a, DAVID KEGG pathway analysis of down-regulated DEGs in

WT-HFD/D6KO-HFD dataset. b, The down-regulated DEGs in HFD-fed dusp6-deficient mice

involved in KEGG cytokine-cytokine receptor interaction pathway. c, qRT-PCR analysis of Ltα,

Ltβ and LTβR of WT and dusp6-deficient mice before and after HFD treatment. For (c), data

are presented as the mean ± SEM. Asterisk (P<0.05) and double asterisks (P<0.005) indicate

statistical difference according to One-Way ANOVA analysis and Tukey post-hoc test. The

number of mice for each group: WT-CD, N=7; WT-HFD, N=8; Dusp6KO-CD, N=7;

Dusp6KO-HFD, N=8.

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Supplementary Figure 5. Dusp6-deficiency reversed HFD-mediated induction of

intestinal AMPs. a, qRT-PCR analysis of intestinal TJP-1 gene of WT/dusp6-deficient mice

before and after HFD treatment. Data are presented as the mean ± SEM. ns indicates

statistical non-significant according to One-Way ANOVA analysis and Tukey post-hoc test.

N=5 for each group. b, Serum endotoxin levels of WT/dusp6-deficient mice before and after

HFD treatment were determined by LAL test. Data are presented as mean ± SEM. Double

asterisks indicate statistical difference (P<0.005) according to One-Way ANOVA analysis and

Tukey post-hoc test. The number of mice for each group: WT-CD, N=9; WT-HFD, N=13;

Dusp6KO-CD, N=9; Dusp6KO-HFD, N=13. c-d, Quantitative RT-PCR validation of RegIIIβ (c)

and RegIIIγ (d). Data are presented as the mean ± SEM. The number of mice for each group:

WT-CD, N=10; WT-HFD, N=8; Dusp6KO-CD, N=9; Dusp6KO-HFD, N=7. ns indicates

statistically non-significant according to One-Way ANOVA analysis and Tukey post-hoc test.

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Supplementary Tables Supplementary Table 1. Taxonomic composition at phylum and family level of fecal/gut

microbiota of WT and dusp6 KO mice before and after HFD treatment.

Dusp6

KO-Chow

WT-Chow Dusp6

KO-HFD

WT-HFD

Samples 11 13 6 6

Good’s coverage

(phylum)

98.22% 98.5% 98.48% 98.83%

Shannon index

(phylum)

6.78 6.78 6.18 6.57

PERMANOVA

/Adonis test

Chow diet fed (WT vs. Dusp6 KO)

R2=0.14841, p=0.002

HFD fed (WT vs. Dusp6 KO)

R2=0.27924, p=0.003

Phylum (%)

Actinobacteria 0.03±0.01 0.06±0.02* 0.24±0.04 0.23±0.05

Bacteroidetes 42.13±6.26 63.75±2.81* 62.94±3.82 44.98±5.27*

Cyanobacteria 0.08±0.01 0.14±0.05 0.00±0.00 0.1±0.03*

Deferribacteres 3.62±1.01 0.83±0.24** 1.05±0.42 1.86±1.32

Firmicutes 53.2±5.64 33.97±2.79* 34.62±3.58 47.70±2.11*

Proteobacteria 0.59±0.08 0.92±0.10* 1.01±0.29 5.08±2.30

TM7 0.02±0.00 0.02±0.01 0.00±0.00 0.01±0.00

Tenericutes 0.35±0.09 0.30±0.09 0.13±0.05 0.03±0.01

Family (%)

Bacteroidaceae 2.24±0.91 1.18±0.23 29.42±3.52 10.98±1.63**

S24-7 27.33±5.95 52.13±3.26** 21.90±1.84 18.70±2.51

Rikenellaceae 9.81±1.45 7.30±0.95 4.85±1.07 6.27±0.95

Porphyromonadaceae 0.07±0.01 0.11±0.03 2.74±0.60 2.59±0.79

Odoribacteraceae 1.99±0.36 2.17±0.35 2.22±0.43 5.24±1.46

Ruminococcaceae 10.30±1.14 6.34±0.90* 11.65±2.21 15.62±1.62

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Erysipelotrichaceae 0.24±0.07 0.18±0.04 8.21±1.79 19.09±1.70**

Lachnospiraceae 12.43±1.85 6.53±0.97** 3.23±0.62 3.84±0.39

Lactobacillaceae 2.72±0.87 5.73±1.59 0.95±0.29 0.846±0.34

Clostridiaceae 1.22±0.35 0.82±0.30 0.055±0.02 0.04±0.02

Data are presented as the mean ± SEM. Asterisk (P<0.05) and double asterisks (P<0.005)

indicate statistical difference according to Mann-Whitney analysis performed on the

comparison of Chow diet fed WT vs. Dusp6 KO mice or HFD fed WT vs. Dusp6 KO mice data

set.

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Supplementary Table 2. Taxonomic composition at phylum and family level of fecal/gut

microbiota of WT and D6KO microbiota recipient mice before and after HFD treatment.

Dusp6

KO-Chow

WT-Chow Dusp6

KO-HFD

WT-HFD

Samples 16 16 8 8

Good’s coverage

(phylum)

99.15% 99.2% 99.06% 99.13%

Shannon index

(phylum)

6.07 6.17 6.56 6.71

PERMANOVA

/Adonis test

Chow diet fed FMT (WT vs.

D6KO) R2=0.11135, p=0.005

HFD fed FMT (WT vs. D6KO)

R2=0.11582, p=0.065

Phylum (%)

Actinobacteria 0.49±0.06 0.47±0.05 0.31±0.06 0.38±0.07

Bacteroidetes 45.12±3.25 45.19±2.63 31.65±2.48 29.88±2.90

Cyanobacteria N.D. N.D. 0.05±0.02 0.03±0.01

Deferribacteres 0.86±0.19 0.92±0.23 0.46±0.14 0.82±0.42

Firmicutes 50.37±3.27 51.55±2.53 66.86±2.50 68.42±2.74

Proteobacteria 3.13±0.54 1.84±0.35 0.40±0.10 0.39±0.08

TM7 0.01±0.00 0.02±0.00** 0.01±0.00 0.02±0.00

Tenericutes 0.02±0.00 0.02±0.00 0.21±0.06 0.07±0.01

Family (%)

Bacteroidaceae 22.74±2.62 22.59±2.42 5.52±0.69 5.71±1.62

S24-7 15.88±0.97 17.67±1.32 16.19±0.74 15.92±0.89

Rikenellaceae 1.44±0.19 1.43±0.18 3.47±0.67 3.57±0.59

Porphyromonadaceae 4.48±0.61 2.97±0.35 0.78±0.15 0.80±0.17

Odoribacteraceae 0.17±0.05 0.29±0.07 5.32±1.55 3.48±0.51

Ruminococcaceae 6.69±0.77 7.90±0.90 13.39±1.67 13.14±2.00

Erysipelotrichaceae 21.03±2.36 26.16±2.50 30.46±5.52 27.64±4.37

Lachnospiraceae 3.58±0.36 3.39±0.36 3.28±0.25 4.74±0.95

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Lactobacillaceae 13.09±1.89 8.32±1.25 6.42±1.32 9.22±2.02

Clostridiaceae 0.07±0.02 0.09±0.01 0.05±0.01 0.06±0.00

Dehalobacteriaceae 0.21±0.03 0.22±0.03 0.21±0.03 0.19±0.03

Enterococcaceae 0.04±0.01 0.10±0.03 0.27±0.11 0.46±0.16

Turicibacteraceae 0.00±0.00 0.06±0.03** 0.08±0.03 0.19±0.05

Peptococcaceae 0.02±0.01 0.02±0.01 0.13±0.04 0.16±0.04

Streptococcaceae 0.05±0.01 0.11±0.02 0.06±0.01 0.17±0.05**

Desulfovibrionaceae 0.21±0.03 0.24±0.05 0.31±0.07 0.23±0.05

Helicobacteraceae 0.06±0.01 0.07±0.014 0.07±0.03 0.14±0.05

Alcaligenaceae 2.63±0.44 1.54±0.34 0.01±0.00 0.00±0.00

Enterobacteriaceae 0.22±0.13 0 0.01±0.00 0.00±0.00*

Data are presented as the mean ± SEM. Double asterisks (P<0.005) indicate statistical

difference according to Mann-Whitney analysis performed on the comparison of Chow diet fed

WT vs. D6KO FMT recipient mice or HFD fed WT vs. D6KO FMT recipient mice data set. N.D.:

Non-Detectable.

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See the supplemented excel file for Supplementary Table 3.

Supplementary Table 4.

qRT-PCR primers for mouse genes and qPCR primers for bacteria

Gene name Direction Sequence Reference

TBP1 Forward 5'-caaacccagaattgttctcctt-3'

1 Reverse 5'-atgtggtcttcctgaatccct-3'

Pparg Forward 5'-gtgccagtttcgatccgtaga-3'

2 Reverse 5'-ggccagcatcgtgtagatga-3'

Adipoq Forward 5'-ccatctggaggtgggagac-3'

Reverse 5'-ctgcatagagtccattgtggtc-3'

Pck1 Forward 5'-tgctgcccttatcattaggttt-3'

3 Reverse 5'-gggtgcagaatctcgagttg-3'

CD36 Forward 5'-ggagcaactggtggatggtt-3'

3 Reverse 5'-ttgagactctgaaaggatcagca-3'

Cyp27a1 Forward 5'-ccacaagggcctcacctatg-3'

4 Reverse 5'-gcacctggtccagccgggtg-3'

Scd1 Forward 5'-ccgggagaatatcctggttt-3'

3 Reverse 5'-cactggcagagtagtcgaagg-3'

Me1 Forward 5'-ctcataggagttgctgcaattgg-3'

3 Reverse 5'-cgttgaaggcagccatatcc-3'

Fabp4 Forward 5'-aaggtgaagagcatcataaccct-3'

Reverse 5'-tcacgcctttcataacacattcc-3'

Angptl4 Forward 5'-acggccaatgagctggg-3'

Reverse 5'-gggcagggaaaggcca-3'

Cldn1 Forward 5'-actccttgctgaatctgaacagt-3'

Reverse 5'-ggacacaaagattgcgatcag-3'

Jam2 Forward 5'-tgtttgtactacatgcgaacctg-3'

Reverse 5'-tccatttccagggagtctga-3'

Jam3 Forward 5'-ggctgcgacttcgactgta-3'

Reverse 5'-gagattcactgcctctatcatgc-3'

Ppm1j Forward 5'-tctctcgacccacctttctg-3'

Reverse 5'-ttgccagcattgatgacct-3'

Pard6g Forward 5'-tcggctatgctgatgtgc-3'

Reverse 5'-gctgtaatggtctgcttcttctc-3'

Myh10 Forward 5'-tgctggttttgaaatttttgag-3'

Reverse 5'-tgccctctcgctggtact-3'

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Gnai1 Forward 5'-acgattcggcagcgtactat-3'

Reverse 5'-atcctgctgagttgggatgt-3'

Rras Forward 5'-tcacaagctggtggtcgtag-3'

Reverse 5'-tgggatcatagtcagacacaaag-3'

Mras Forward 5'-ctctcctgtgccgagtgg-3'

Reverse 5'-actcctcagggctttggtct-3'

Lta Forward 5'-cacactgccgcttcctctat-3'

5 Reverse 5'-gccgagcagtgtcatgtg-3'

Ltb Forward 5'-cctggtgaccctgttgttg-3'

5 Reverse 5'-tgctcctgagccaatgatct-3'

LtbR Forward 5'-ccataccagatgtgagatccag-3'

Reverse 5'-atggccagcagtagcattg-3'

TJP1 Forward 5'-atgcagacccagcaaagg-3'

Reverse 5'-tggttttgtctcatcatttcttca-3'

Defa5 Forward 5'-agctggctgactgggtgtgt-3'

6 Reverse 5'-cggtgcttcggtctccacgg-3'

RegIIIb Forward 5'-atggctcctactgctatgcc-3'

Reverse 5'-gtgtcctccaggcctcttt-3'

RegIIIg Forward 5'-atggctcctattgctatgcc-3'

7 Reverse 5'-gatgtcctgagggcctctt-3'

Eubacteria UniF340 5'-actcctacgggaggcagcagt-3' 8 UniR514 5'-attaccgcggctgctggc-3'

Akkermansia

muciniphila

Forward 5'-cagcacgtgaaggtggggac-3' 9

Reverse 5'-ccttgcggttggcttcagat-3'

SFB SFB736F 5'-gacgctgaggcatgagagcat-3'

10 SFB844R 5'-gacggcacggattgttattca-3'

15

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